The publications and reference materials noted herein are each incorporated by reference in their entirety.
The hyaluronidase enzyme family consists of enzymes capable of hydrolyzing or “breaking down” the polysaccharide hyaluronic acid. Hyaluronic acid is an important constituent of connective tissue. Thus, hyaluronidases, which can spread and diffuse rapidly through tissues, can modify the permeability and viscosity of the intercellular cement by hydrolyzing hyaluronic acid.
Hyaluronidases can be found in various animal tissues, e.g., mammalian testicular and spleen tissue, and in snake venom, and in certain species of Streptococcus and Staphylococcus. Basically, the enzyme family comprises three major groups: the testicular type (hyaluronoglucoronidase) (International Union of Biochemistry and Molecular Biology (IUMB) No. EC 3.2.1.35); the leech type (hyaluronoglucoronidase) (IUMB E.C. 3.2.1.36); and the bacterial type (hyaluronate lyase) (IUMB E.C. 4.2.2.1). Between these groups, there are chemical and biological differences in, for example, substrate specificity, optimum pH, stability in aqueous and non-aqueous solutions, and thermal stability. For example, many bacterial hyaluronidases are only active towards hyaluronic acid or hyaluronate while testicular hyaluronidases hydrolyses both hyaluranic acid and other mucopolysaccharides (chondroitin, chondroitin sulfate). However, there are even greater variations within each group, in part related to the source of the enzyme.
Various hyaluronidases and methods for preparing and using them are known in the art (Linker A., Hyaluronidase. In: Methods of enzymatic analysis, Eds. Bergmeyer H U. Bergmeyer J, Grasl M, Verlag Chemie Gmbh. Weinheim, 1984, pp. 256-262; King T P, Spangfort M D, Int Arch Allergy Immunol. 2000 October; 123(2):99-106; Jedrzejas M J, Crit. Rev Biochem Mol Biol, 2000; 35(3):221-51: Hynes W L. Walton S L, FEMS Microbiol Lett. 2000 Feb. 15; 183(2):201-7; Menzel F J, Farr C; Cancer Lett 1998; 131(1):3-11). For example, U.S. Pat. No. 4,258,134 relates to hyaluronidase from Streptomyces koganeiensis, having optimal activity at about pH 4.0; and Japanese Patent Nos. 63044883 and 62104579 provide hyaluronidase from Streptococcus dysgalactiae, the enzyme having a molecular weight of about 80 kD, and an optimum pH range of 5.8 to 6.6, which is inhibited by Fe2+ and Cu2+. Russian Federation Patent No. 2005488 describes a Streptomyces actinocidus hyaluronidase preparation termed Actinogial, which has a pH optimum of about 6.5 and a specific activity of about 30-40 IU/mg. Regarding hyaluronidase from non-bacterial origin, U.S. Pat. Nos. 4,904,594 and 5,061.627 relate to preparations of hyaluronidase from krill and other crustaceans: U.S. Pat. No. 5,593,877 is concerned with hyaluronidases and other proteins from vespid venom nucleic acids; U.S. Pat. Nos. 5,747,027 and 5,827,721 describe purified hyaluronidase of mammalian origin; and U.S. Pat. No. 5,854,046 and PCT publication WO 99/29841 are directed to human hyaluronidases.
Methods for producing and purifying hyaluronidases from various sources have been developed, as described in, e.g., U.S. Pat. No. 4,410,531, disclosing a method for purifying an enzyme from a crude hyaluronidase preparation; Swiss Patent No. CH628088, providing a method for purifying hyaluronate lyase and other proteins from Streptococci cultures; and U.S. Pat. No. 1,060,513, describing a method for preparing hyaluronidase from animal organs. See also SU1723121. U.S. Pat. No. 4,897,349 provides a method for increasing microbial biosynthesis of hyaluronidase by regulating oxygen concentration. Various types of hyaluronidase can be obtained commercially, e.g. from Wyeth-Ayerst (Wydase®), Abbot (Hyazyme), Bristol-Myers Squibb (Enzodase), and Ortho Pharmaceuticals (Diffusin).
One useful property of hyalurodinase is that it can reduce formation of scar tissue of varying etiologies and “soften” the skin. For example, methods for dissolving mammalian scar tissue by administering hyaluronidase and collagenase into the lesion are disclosed in U.S. Pat. Nos. 4,524,065 and 4,645,668; and methods for improving skin penetration of various topically applied drugs have been suggested by PCT publications WO 00/38732; WO 01/45743: and German Patent No. 19963538. Topically or parenteral administration of hyaluronidase can also be used to promote diffusion of substances, whether applied topically or injected. This has been applied clinically, where hyaluronidases have been used to facilitate the distribution of drugs or biological agents, usually in skin (Nara et al., Chem Pharm Bull (Tokyo) 1992; 40:737-40: Costello and Jeske, Phys Ther 1995; 75:554-63; Laugier. Br J Dermatol 2000 February; 142(2):226-33).
Hyaluronidases have also shown useful in a wide range of other medical applications, including blocking lymph node invasion of tumor cells (PCT publication WO 95/30439); treatments of vascular diseases (German Patent No. 19860541 and U.S. Pat. No. 4,568,543) and prostatic hypertrophy (U.S. Pat. No. 5,116,615): vaccination against helminth infection (U.S. Pat. No. 5,811,100); and for various ophthalmologic applications (U.S. Pat. Nos. 5,292,509; 5,856.120, and 5,866,120). For example, hyaluronidase has been used to reduce intraocular pressure in the eyes of glaucoma patients through degradation of hyaluronan within the vitreous humor (U.S. Pat. No. 4,820,516).
Hyaluronidase has also been used in cancer therapy as a “spreading agent” to enhance the efficacy of chemotherapeutics and/or the accessibility of tumors to chemotherapeutics (Schuller et al., Proc. Amer. Assoc. Cancer Res. 1991; 32:173, abstract no. 1034; Czejka et al., Pharmazie 1990:45:H.9) and has been used in combination with other chemotherapeutic agents in the treatment of a variety of cancers including urinary bladder cancer (Horn et al., 1985, J. Surg. Oncol., 2:304-307), squamous cell carcinoma (Kohno et al., 94, J. Cancer Res. Oncol., 120:293-297), breast cancer (Beckenlehner et al., 1992, J. Cancer Res. Oncol. 118:591-596), and gastrointestinal cancer (Scheithauer et al., 1988, Anticancer Res. 8:391-396). Administration of hyaluronidase also induces responsiveness of previously chemotherapy-resistant tumors of the pancreas, stomach, colon, ovaries, and breast (Baumgartner et al., Reg. Cancer Treat. 1988; 1:55-58; Zanker et at., Proc. Amer. Assoc. Cancer Res. 1986; 27:390). In addition, serum hyaluronidase prevents growth of tumors transplanted into mice (De Maeyer et al., Int. J. Cancer 1992; 51:657-660), while injection of hyaluronidase inhibits tumor formation caused by exposure to carcinogens (Pawlowsli et al., Int. J. Cancer 1979; 23:105-109; Haberman et al., Proceedings of the 17th Annual Meeting of the American Society of Clinical Oncology, Washington, D.C., 1981; 22:105, abstract no. 415). Intravenous or intramuscular injection of hyaluronidase is also effective in the treatment of brain cancer (gliomas) (PCT published application No. WO88/02261).
While there is a vast number of existing and potential therapeutic and cosmetic applications for hyaluronidase, a remaining problem is that currently available hyaluronidases are usually chemically and thermally unstable, often have significant non-specific activity, and show peak activity at an acidic pH, thus having suboptimal properties for in vivo applications. There is therefore a need in the art for hyaluronidase preparations that are stable in various formulations, and that show a high stability and activity at physiological temperatures and pH. The present invention addresses these and other needs in the art.